The Length of N-Glycans of Recombinant H5N1 Hemagglutinin Influences the Oligomerization and Immunogenicity of Vaccine Antigen

Abstract

Hemagglutinin glycoprotein (HA) is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm) and with low-mannose glycosylation (H5Man5) were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.

Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison

“…In most cases, only shake-flask experiments were performed, in which comparisons to other strains or impact of product glycosylation patterns can be biased, due to the uncontrolled behavior in terms of process control (e.g. pH, dissolved oxygen) [ 19 , 20 , 22 , 23 ]. In literature, environmental stressors are known to affect a protein’s post-translational processing [ 24 ], which highlights the importance of analyzing a protein’s properties during the controlled production in bioreactors.…”

High-Titre Neutralizing Antibodies to H1N1 Influenza Virus after Mouse Immunization with Yeast Expressed H1 Antigen: A Promising Influenza Vaccine Candidate

“…Previously, we showed that immunization with the subunit vaccine based on the extracellular region of the H5 haemagglutinin with deletion of the multibasic cleavage site elicited serum haemagglutination-inhibiting antibodies and fully protected chickens from lethal infections by the highly pathogenic H5N1 virus [9]. We also demonstrated the feasibility of producing H5N1 HA antigen in yeast [9, 10]. Therefore, testing this expression system for production of other influenza antigens, especially those HAs which are components of strains typical for humans, was successful.…”

“…The crucial issue for a vaccine to be licensed for medical use is to develop an effective process for the purification of the protein product [10]. Our work addresses both vaccine production steps: the expression and the purification.…”

“…As the influenza virus neutralizing antibodies currently are directed primarily against the haemagglutinin, recombinant HA-based vaccines provide a promising alternative for influenza vaccine manufacture. Such a vaccine comprises a recombinant haemagglutinin obtained by genetic engineering using various expression systems [4–10].…”

“…The aim of this study was to test an H1N1pdm09 influenza virus HA produced in a yeast expression system as a potential vaccine antigen. Our previous study showed that the H5 antigen produced in the P. pastoris cells is capable of inducing a specific immune response in mice [8, 10] and providing full protection in chicken [9]. Ease of preparation, low cost of production, and high immunogenicity of the yeast-derived antigen prompted us to test an H1N1pdm09 influenza virus antigen.…”